A potentially catastrophic influenza pandemic is inevitable. Further, technology now allows the laboratory creation of highly pathogenic influenza viruses. Preparedness is vital, and it requires optimal use of the available vaccines and antivirals. Although vaccines offer the best control of influenza, at least 6 months is needed for production of a strain-specific vaccine. Therefore, antiviral drugs may be the best immediately available strategy. We hypothesize that combination therapy with two classes of anti-influenza drugs that target different viral proteins and exert different mechanisms will offer clinical and strategic advantages. This hypothesis will be tested by three specific aims. 1) To establish a reliable assay method, we will first evaluate the suitability of newly developed MDCK-SAIT1 cells, which have increased expression of humanlike sialic acid (alpha2-6 Gal-linked) receptors, for detecting drug sensitivity of emerging and resistant variants of different subtypes, including highly pathogenic viruses. 2) We will test combinations of NA inhibitors with the M2 blocker rimantadine against influenza A virus infection in MDCK-SAIT1 cells, and we will characterize their mode of interaction (additive, synergistic, or antagonistic) against human and emerging highly pathogenic H5N1 and H9N2 influenza viruses in a mouse model. 3) We will determine the effect of combination antiviral therapy on the emergence of drug-resistant variants and on clinically important properties of resistant strains, such as virulence and transmissibility. We propose to analyze a wide spectrum of influenza viruses isolated from various hosts and differing in pathogenicity. We will include recent isolates from Hong Kong, because of strong experimental evidence that the precursors of human pandemic influenza viruses originate in southern China. These findings will show 1) whether combination antiviral treatment is clinically advantageous as a response to natural or manmade highly pathogenic influenza viruses and 2) whether this strategy offers practical, economic, and public health advantages for the stockpiling and large-scale use of antivirals.